Several pneumococcal polysaccharide-protein conjugate vaccines are currently in clinical trials. These conjugate vaccines have used tetanus toxoid or diphtheria toxoid as the carrier protein. Such vaccines provide protection primarily against capsular types contained in the vaccine Use of pneumococcal protein antigens such as pneumococcal surface protein A (PspA), pneumolysin (Ply) or hemin-binding protein may provide broader immunity, and can be used as carriers for the conjugates to provide an additional protective antigen for prevention of pneumococcal infection. The objective of the present study was to evaluate the antibody response using ELISA to pneumococcal type 9V polysaccharide conjugated to inactivated pneumolysin or autolysin (Aly). Cross-protective immunity induced by conjugates using the pneumococcal proteins, Ply or Aly, was examined by bacterial clearance from blood of mice challenged with various types of virulent pneumococci and by opsonophagocytic activity. Mice injected with type 9V polysaccharide conjugated to inactivated pneumolysin or autolysin produced high levels of specific IgG and IgM antibodies to both the polysaccharide and the protein carrier. Mice immunized with glycoconjugates using pneumococcal protein carriers exhibited rapid bacterial clearance from blood and provided cross-protection against heterologous serotypes of virulent pneumococci. Thus, conjugates using pneumococcal protein carriers can induce opsonophagocytic activity to destroy homologous and heterologous pneumococci, indicating that such conjugates could confer broader protective immunity than conjugates using the toxoid proteins as carriers for prevention of pneumococcal diseases.